Analysis of molecular binding and cell behavior are important for disease diagnostics, biomedical research, and drug discovery. The vast majority of array-based studies of bioaffinity interactions employ fluorescently labeled biomolecules or enzyme-linked colorimetric assays. However, there is a need for methods that detect bioaffinity interactions without molecular labels, especially for biomolecular and cellular interactions, where labeling is problematic and can interfere with their biological properties. The development of simple and specific biosensors to detect biomarkers and measure cellular response has far-reaching implications in their timely detection which is of great concern to human health and safety.
The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. The antibody and nucleic acid fluorescence-based detection approaches currently consist of complex, multi-step, time consuming, and labor intensive assay formats and target analyte analysis to ensure the specificity of detection. Additionally, these methods are not suitable for the rapid pathogen or cancer detection as they require extensive blood culture of the pathogen or diseased tissue in the central laboratory prior to the detection of antibodies.
The analysis of bio-molecular interactions is also a key part of the drug discovery process which involves determining the binding affinity of the drug to the target protein of interest. Even though developments in the field of high-throughput screening (HTS) and computational chemistry greatly accelerated and facilitated the drug finding process, there are significant limitations to overcome. An example is the fluorescence-based HTS assay, which may generate false positive (e.g. binding to the reporter enzyme or direct hydrophobic interaction of the label with the target) or false negative results (e.g. occluding of the binding site). The application of novel and efficient label-free technologies is of high importance to the drug discovery process, since they will lower development costs and decrease the time to market.
For drug discovery as well as in the biomedical research, the study of the effect of the specific cues (e.g. chemical, topographical, flow, etc.) on cell attachment and motility, cell viability, cell proliferation and cell cycle is of paramount importance. Inducing and subsequent measurement of a specific cellular response requires providing the cells with the appropriate cues, to control the conditions in the cell microenvironment, and to monitor cellular responses on multiple hierarchical levels within a large number of parallel experiments. Currently employed assays that rely on cell culture in Petri dishes and subsequent fluorescence-based live-cell imaging and biomolecule detection are slow, cumbersome and cannot meet these requirements. Multi-well plate assays can increase the throughput through automatic imaging afforded by high content cell screening (HCCS). However, an important consideration for the multi-well assays is ensuring uniform patterning or treatment of each well which is often precluded by variations in the volume of liquid dispensed into each well. The resulting variability in the concentration of applied reagents hinders fair and quantitative comparisons and limits the ability of HCCS to resolve small differences in cell signaling responses. This issue is exacerbated in more complex protocols, such as sequential exposure of cells to different media, because of errors that accumulate when changing media. Moreover repeated media aspirations might unintentionally remove cells from the wells. Because these assays are also difficult to miniaturize, HCCS experiments may consume large quantities of expensive or valuable cells and reagents. Finally HCCS still relies on fluorescent tags which may trigger unwanted steric hindrance effects.
Consequently, the research into the effect of cues (single or multiple) on cellular response to date has been limited by the lack of robust and reproducible methods for homogeneous material production, precise control of the cell culture conditions and in situ real-time label-free monitoring of cellular response, cell behavior, cell viability or biomolecular binding interactions. Specifically, the material production methods have lacked the control required to reproducibly fabricate homogeneous surfaces that will allow investigations into specific interactions between cells and isolated variables i.e. a precisely defined nanoscale patterns in a defined space with control over the induced change in topography and associated changes to surface energy. The commonly employed well-based cell culture methods are costly and suffer errors in liquid dispensing, both manually and robotically, thus precluding uniform handling of each well which in turn limits how finely signaling responses may be resolved. Finally, while the use of fluorescent imaging techniques for cell analysis can provide information not easily attainable by other methods, they are usually confounded by the need to over-express the signaling protein of interest and by possible effects of the fluorescent marker on the protein's function. Therefore due to the phenomenological nature of current studies, the responses achieved have been heterogeneous at both single cell and cell population levels (Balasundaram 2007; Barbucci 2003; Blümmel 2007; Curtis 2006; Dalby 2007a; Ernsting 2007; Kimura 2007; Salber 2007).
Fluorescence and chemiluminescent detection are the most common methods employed for biomolecule recognition. Both schemes require the use of a labeled recognition element which binds to a molecule of interest thus producing a selective signal upon binding (Marquette 2006). Currently, the detection and quantification of genomic and proteomic biomarkers from serum or other physiological samples rely on solid-phase detection, where strong amplification chemistry is often needed to produce a readout. In the case of DNA markers, the state of the art relies on polymerase chain reaction (PCR), while for the protein markers enzyme-linked immunosorbent assay (ELISA) prevails. Many attempts at miniaturizing bench-top systems using microfluidics in order to increase the detection limits and reduce incubation times, reagent consumption and sample size have been reported with impressive results (Zhang 2009a; Zhang 2009b; Lim 2007; Lee 2006; Malic 2007). However, despite a growing focus from the microfluidic research community, both PCR and ELISA rely on fluorescence labels, which increase the complexity and cost of the assay. In addition to the requirement for a labeled recognition element, these techniques typically require complex optical systems which typically consist of a large microscope or a microplate reader. As a result, the field of microfluidics has yet to produce many commercial devices for disease diagnostics (Myers 2008). There is a need for coupling and integrating microfluidics with direct label-free detection methods that base themselves on physical characteristics of biologic phenomena and have the potential to reduce reagent costs and test complexity (Weigl 2008).
The development of minimally invasive techniques to induce a specific cellular response is focused on controlling the direct contact and interaction between a given cell type and a well defined material. One way of controlling cell adhesion and subsequent morphology is by nanotopography. Research has shown that cells can detect and respond to an array of topographies and can be affected by the level of order of an induced topography, with clear effects on cell functionality (Dalby 2009; Dalby 2008; Dalby 2007b; Dalby 2007c; Dalby 2007d; Hochbaum 2010). Similarly, bacteria also respond to topographical (spatial and mechanical) cues and spontaneous bacteria patterning on a periodic nanostructure array has recently been shown (Hochbaum 2010). Another method employs chemically modified surface to induce cellular response (Cavalcanti-Adam 2008). For both strategies to prove successful the material must be homogenous, robust and fabricated or functionalized in a reproducible manner. To date, several methods have been used for this purpose, including electron-beam lithography, nanoimprint lithography and dip-pen nanolithography (Cavalcanti-Adam 2007; Curran 2010). However, the assays in these studies were performed using traditional cell-culture methods and analyzed using live fluorescence microscopy with inherent drawbacks of these techniques which may have resulted in misleading interpretation of results due to error in liquid handling, perturbation caused by fluorescent markers and low throughput in which only a few cells were imaged for each experimental run.
Flow cytometry (FC) and laser scanning cytometry (LSC) are the most widely used techniques for cell analysis with well characterized distributions of cellular behaviour. Both techniques use fluorescent dyes to label biomolecules of interest within the cell in order to reveal the information about the quantity of biomolecules within the cell. Flow cytometry involves the hydrodynamic isolation of individual cells thus affording high throughput serial analysis. However, FC is limited to characterizing fluorescent signals (GFP-fusion proteins, immunofluorescence, and fluorogenic substrates to intracellular enzymes) (Fayet 1991; Nolan 1998; Krutzik 2006), which can lead to steric hindrance and is incapable of important time-dependent measurements of the cell population. Conversely, laser scanning cytometry (LSC) relies on the use of a scanning laser to excite the dyes on surface immobilized cells (Griffin 2003; Bedner 1993) thereby allowing kinetic measurement of time-dependent information in individual cells. However, only a limited region of a plate can be scanned thus limiting the throughput of the technique. Additionally, the introduction of reagents is performed using a pipette and only slow time dependent changes after solution exchange are meaningful. This is particularly due to uneven introduction of solution over the whole slide or plate, and the serial process of laser scanning. Furthermore, cells analyzed using both methods are usually grown in traditional flasks, slides or Petri-dishes before analysis, and so uniformity of environment is limited to that of the flask or dish. Notably, cell-cell contact is not controllable, and diffusible secretions are maintained in the culture environment.
To overcome some of these limitations, research has recently shifted towards the exploitation of the precise chemical delivery capabilities of microfluidic devices. The single most popular approach for the fabrication of microfluidic devices for cell-based assays is based on the soft-lithography of polydimethylsiloxane (PDMS). PDMS is an elastomer which is casted over a mold typically fabricated using photolithography and cured for several hours resulting in a transfer of features from the mold to the PDMS. Its wide use as a material of choice is due to its mechanical property, which is amenable to integration of fluidic valves, essential elements for major microfluidic applications. PDMS platforms for cell culture have been reported in the past especially for two-dimensional morphological cells, such as epithelial cells, and several designs have been the subject of patent applications (Jin 2010; Lee 2010). However, most of these studies coupled microfluidic device to traditional macroscale equipment (i.e. fluorescence microscopes) and relied on the use of fluorescence imaging for cellular response analysis.
Moreover, there is surprisingly little work reported on the combination of nanopatterned surfaces and microfluidics, especially in a way advantageous for studying topographically induced cellular response. This is in part due to the difficulty in reproducibly fabricating nanostructured surfaces within microfluidic cell culture devices. Several production processes have been reported for nanostructure fabrication inside microchannels including vapor deposition of nanoparticles (Song 2009), in situ formation of nanoparticles inside the channels from catalytic reaction (Fonverne 2009), and polymerization of a polymer around an anodic aluminum oxide template (Soper 2008). However, controlling the regularity, geometry and/or spacing of the nanoparticle arrays using these techniques is difficult to achieve limiting the reproducibility of the experimental measurements.
In order to obtain spatially controlled geometry and spacing of the nanostructures within a PDMS microfluidic channel, multilayer mold comprising nano- and micro-structures are required with fabrication procedures involving sequential electron-beam lithography, interference lithography or nanoimprint lithography in concert with photolithography of SU-8 resist. The compatibility of materials and reagents involved in these processes is difficult to achieve. Additionally, once the mold is fabricated and the microchannels have been defined, the slightest change of the microfluidic layout would require the repetition of complete fabrication process, starting with the nanostructured substrate. This can result in topographical surface variations induced by sample-to-sample fabrication differences. Furthermore, PDMS soft-lithography fabrication technique itself is not well suited for mass production of microfluidic devices which hinders their application in industry, including medical diagnosis and pharmaceutics. Finally, the use of PDMS as a material for in vitro models for cell culture needs to be considered in a biological context due to the leaching of uncured oligomers from the polymer network into microsystem media (Regehr 2009).
Further, microfluidic devices with nanostructured hydrophobic surfaces have been developed to control surface tension and liquid pressure in fluid flow channels (Extrand 2005), but the standard techniques used to nano-pattern the channel surfaces are insufficiently flexible to permit simple and fast patterning of nanostructures, especially different nanostructured patterns, at specific locations in the channels or chambers of the device but not at others. Thus, different design features within the same device are difficult to accomplish and the designs are difficult to adapt to the requirements of plasmonic detection techniques.
In general, prior art systems have one or more deficiencies. There is a lack of an integrated microfluidic system that relies on non-invasive, label-free detection technologies including plasmonic techniques such as surface plasmon resonance (SPR) (e.g. reflection-mode SPR, transmission-mode SPR, localized surface plasmon resonance (LSPR)) and surface-enhanced Raman spectroscopy (SERS) for monitoring cell behavior, cell-substrate interactions, cell response to stimuli and biomolecule detection. There is a lack of fabrication techniques allowing monolithically integrated nanostructured cell culture system in long-term biocompatible materials with simultaneous cell guiding functionality and plasmonic detection capability using topographical cues and nanostructure plasmonic response, respectively. There is poor control of cellular microenvironment in Petri-dish or microwell plates. There is lack of reproducible and robust surface topography (nanopatterning) for precise control of cellular response and cell-substrate interaction studies. There is a lack of integrated nanostructured surface within microfluidic channels. And, there is a lack of low-cost and rapid mold fabrication techniques that allow interchangeable nano- and micro-structure design.
There remains a need for an integrated system that can meet one or more of the following requirements: (i) efficient control of initial cell adhesion; (ii) efficient control of the cellular response to the specific stimulus over a prolonged period; (iii) in situ, label-free and real-time monitoring of cellular response, cell mobility, cell behavior, cell-viability or biomolecule detection in order to avoid false response due to cellular secretion of the molecules to which they respond and steric hindrance induced by the fluorescent tags. Additionally, the system is ideally low-cost, portable and amenable to mass-production.